1. Field of the Invention
This invention relates to the field of polypeptides having antimicrobial activity and the polynucleotides encoding them. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid constructs. The invention more specifically relates to an antimicrobial phage-associated HydH5 peptidoglycan hydrolase polypeptide, truncations of the HydH5 peptidoglycan hydrolase polypeptide, and fusion polypeptides comprising the HydH5 peptidoglycan hydrolase and truncated HydH5 polypeptides. This invention also relates to synergistic pathogen-specific compositions comprising the endolysin LysH5 together either with HydH5 or with fusion polypeptides comprising HydH5. The invention further relates to compositions and methods of making the polypeptides and methods of treating staphylococcal-associated diseases, including methicillin-resistant Staphylococcus aureus (MRSA).
2. Description of the Relevant Art
Staphylococcus aureus is a notorious pathogen that causes numerous pathologies including food poisoning, toxic shock syndrome, endocarditis, and skin and wound infections, (Lowy, F. D. 1998. N. Engl. J. Med. 339:520-532). The emergence of multidrug-resistant strains, especially methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) is raising serious concerns due to their high frequency in both nosocomial and community-acquired settings (Kock et al. 2010. Euro Surveill. 15(41):19688).
Recent results with phage therapy in animal models have driven much interest in phages and phage-encoded proteins to treat infections (O'Flaherty et al. 2009. FEMS Microbiol. Rev. 33(4):801-819; Fenton et al. 2010. Bioeng. Bugs. 1(1):9-16). These studies clearly show the efficacy of phages and lysins in killing human pathogenic bacteria in animal models (Matsuzaki et al. 2005. J. Infect. Chemotherapy 11:211-219; Fischetti, V.A. 2010. Int. J. Med. Microbiol. 300(6):357-362). Specifically, several assays have been performed against S. aureus bacteremia. The intraperitoneal administration of phages phiMR11 and phiMR25 rescued mice inoculated with a lethal dose of S. aureus (Matsuzaki et al. 2003. J. Infect. Dis. 187(4):613-624; Hoshiba et al. 2010. Arch. Virol. 155(4):545-552). Moreover, in a rabbit model of wound infection caused by S. aureus, phages prevented abscess formation (Wills et al. 2005. Antimicrob. Agents Chemother. 49(3):1220-1221). Phage lytic proteins also showed successful results. The intraperitoneal administration of the endolysin MV-L from phage phiMR11 protected mice against S. aureus MRSA septic death (Rashel et al. 2007. J. Infect. Dis. 196(8):1237-1247). In another animal model, bacteremia in unprotected mice reached colony counts of ˜107 cfu/ml within 3.5 h after challenge, whereas the administration of the lytic enzyme LysGH15 1 h after MRSA injection was sufficient to protect mice with the mean colony count being less than 104 cfu/ml (Gu et al. 2011. J. Clin. Microbiol. 49(1):111-117). Furthermore, the activity of phage lytic proteins may be increased by using them in combination with other antimicrobials. Both in vitro (Becker et al. 2008. FEMS Microbiol. Lett. 287(2):185-191; Daniel et al. 2010. Antimicrob. Agents Chemother. 54(4):1603-1612; García et al. 2010. Int. J. Food Microbiol. 141(3):151-155) and in vivo synergy (Daniel et al., supra) between phage endolysin constructs and antibiotics or bacteriocins against S. aureus have been reported.
Bacterial cell walls of both Gram-positive and Gram-negative bacteria are composed of peptidoglycan, a complex molecule with a sugar backbone of alternating N-acetylglucosamine and N-acetyl muramic acid residues cross-linked with peptide bridges. Peptidoglycan prevents osmotic lysis of cell protoplast and confers rigidity and shape on cells. Peptidoglycan hydrolases are essential for modifying the peptidoglycan to allow the cell to grow and divide (Vollmer et al. 2008. FEMS Microbiol. Rev. 32(2):287-306). There are three major peptidoglycan hydrolase activities, namely, (i) glycosidase, (ii) amidase, and (iii) endopeptidase activities. Most peptidoglycan hydrolases are composed of a C-terminal cell wall-binding (CWB) domain and a N-terminal catalytic domain(s) (Fischetti, V.A. 2005. Trends in Microbiol. 13:491-496). Bacteriophages also encode peptidoglycan hydrolases (Hermoso et al. 2007. Curr. Opin. Microbiol. 10(5):461-472) that play essential roles in the phage life cycle to allow both entry (virion-associated peptidoglycan hydrolases) and release (endolysins) of the mature phage particles.
In addition to endolysins, other phage encoded proteins (virion-associated peptidoglycan hydrolases) have a potential as antimicrobials. Some bacteriophage virions harbor virion-associated peptidoglycan hydrolases that facilitate the entry of phage DNA across the bacterial cell envelope during infection (Moak and Molineux. 2004. Mol. Microbiol. 51:1169-1183). They are also responsible for the “lysis from without”, a phenomenon caused by some phages when adsorbed onto the host cell at very high numbers (Delbrück, M. 1940. J. Gen. Physiol. 23(5):643-660). This type of peptidoglycan hydrolase activity has been described from a variety of different phage particles infecting S. aureus (Moak and Molineux, supra; Rashel et al. 2008. FEMS Microbiol. Lett. 284:9-16; Takac and Blasi. 2005. Antimicrob. Agents Chemother. 49(7):2934-2940), Lactococcus lactis (Kenny et al. 2004. J. Bacteriol. 186:3480-3491), E. coli (Molineux, I. J. 2001. Mol. Microbiol. 40:1-8; Kanamaru et al. 2002. Nature 415:553-557), and Salmonella (Steinbacher et al. 1997. J. Mol. Biol. 267:865-880).
Recently, a peptidoglycan hydrolase (HydH5) encoded by phage vB_SauS-philPLA88 (philPLA88) has been identified and characterized (Rodríguez et al. 2011, BMC Microbiol. 11: 138). HydH5 has a N-terminal CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) lytic domain and a C-terminal LYZ2 (lysozyme subfamily 2) lytic domain. HydH5 does not have a recognized cell wall binding domain. The full-length 634 amino acid HydH5 and truncated version harboring just one lytic domain (and 6×His-tag) have been overproduced in E. coli. The nickel chromatography purified proteins are able to kill viable S. aureus cells. HydH5 is highly thermostable since it showed antimicrobial activity after heat treatment (100° C., 5 min) (Rodríguez et al., supra).
Lysostaphin is a bacteriocin secreted by S. simulans that lyses S. aureus (Browder et al. 1965. Biochem. Biophys. Res. Commun. 19: 389). The endopeptidase activity is specific to the glycyl-glycyl bonds of the staphylococcal peptidoglycan inter-peptide bridge. It is known that lysostaphin can kill planktonic S. aureus (Walencka et al. 2005. Pol. J. Microbiol. 54: 191-200; Wu et al. 2003. Antimicrob. Agents Chemother. 47: 3407-3414), as well as MRSA (Dajcs et al. 2000. Am. J. Ophthalmol. 130: 544), vancomycin-intermediate S. aureus (Patron et al. 1999. Antimicrob. Agents Chemother. 43:1754-1755), and other antibiotic-resistant strains of S. aureus (Peterson et al. 1978. J. Clin. Invest. 61: 597-609). Lysostaphin can also kill S. aureus growing in a biofilm (Walencka, supra; Wu, supra), and it exhibits limited activity against coagulase-negative staphylococci (Cisani et al. 1982. Antimicrob. Agents Chemother. 21: 531-535); McCormick et al. 2006. Curr. Eye Res. 31: 225-230).
The endolysin LysH5 is encoded by philPLA88 phage and has three putative domains: a cysteine, histidine-dependent amidohydrolases/peptidase (CHAP) domain, an amidase-2 domain, and a C-terminal SH3b cell wall-binding (CWB) domain. LysH5 is able to inhibit the S. aureus growth in milk (Obeso et al. 2008. Int. J. Food Microbiol. 128(2):212-218) and showed a synergistic antimicrobial effect with the bacteriocin nisin (Garcia et al. 2010, supra).
Antibiotic resistance in combination with other important virulence determinants, such as surface-located binding proteins to facilitate adhesion to host tissue, as well as many mechanisms to evade attack by human host defenses makes S. aureus a threatening pathogen (Otto, M. 2010. Ann. Rev. Microbiol. 64:143-162). Novel therapeutic agents specific for staphylococcal species, including methicillin-resistant Staphylococcus aureus (MRSA), are sorely needed to counter the rise of drug resistant pathogenic bacteria.